LOCUS       sequence-380182-        5455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Nimble cloning-compatible vector for protein subcellular 
            localization in plants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5455)
  AUTHORS   Yan P, Tuo D, Shen W, Deng H, Zhou P, Gao X
  TITLE     A Nimble Cloning-compatible vector system for high-throughput gene 
            functional analysis in plants.
  JOURNAL   Plant Commun. 2023 Mar 13;4(2):100471. doi: 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9.
  PUBMED    36352791
REFERENCE   2  (bases 1 to 5455)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Commun."; date: "2023-03-13"; volume: "4(2):100471. doi"; pages: " 
            10.1016/j.xplc.2022.100471. Epub 2022 Nov 9"
COMMENT     Sequence Label: pNC-RFP-N
FEATURES             Location/Qualifiers
     source          1..5455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        184..529
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     472..492
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     CDS             545..1219
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             545..1219
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(675..693)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     943..962
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1137..1160
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     promoter        1401..1503
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1534..1839
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1718..1737
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     terminator      1922..2174
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     primer_bind     complement(2192..2208)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2192..2208)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2245..2265)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2245..2263)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2284..2300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2284..2300)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2297..2319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2308..2324
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2332..2338,2339..2356,2357..2362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2377..2398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2515..2532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2637..2661
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(2752..3340)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2832..2851)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3511..4326)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3511..4326)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4234..4253
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4318..4337
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4617..5052
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
     misc_feature    5179..5201
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     primer_bind     5351..5373
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5365..5382
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5366..5382
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5392..5411
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5392..5410
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     5435..5451
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     5436..5452
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca
       61 gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga
      121 gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc
      181 aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc
      241 tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca
      301 ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg
      361 acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca
      421 agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc
      481 gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggactct
      541 tgacatggcc tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga
      601 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      661 gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
      721 catcctgtcc cctcagttcc agtacggctc caaggcctac gtgaagcacc ccgccgacat
      781 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
      841 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
      901 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
      961 gaccatgggc tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg
     1021 cgagatcaag atgaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac
     1081 cacctacatg gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg acatcaagct
     1141 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg
     1201 ccgccactcc accggcgccc agtggtctct gtccagtcct ggcctcgtcg gccattctcg
     1261 actaagttgg cagcatcacc cgacgcactt tgcgccgaat aaatacctgt gacggaagat
     1321 cacttcgcag aataaataaa tcctggtgtc cctgttgata ccgggaagcc ctgggccaac
     1381 ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa
     1441 ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct
     1501 aaacttttgc tgacgagaac agggactggt gaaatgcagt ttaaggttta cacctataaa
     1561 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcctggg
     1621 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1681 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1741 agtgtgccgg tatccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1801 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     1861 agggccagtc tggccacttg tggtctgctg agaccgtgag gtgaccagct cgaatttccc
     1921 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
     1981 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
     2041 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
     2101 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
     2161 tatgttacta gatcgggcct gcagcccggg ggatccacta gttctagagc ggccgccacc
     2221 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc
     2281 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     2341 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     2401 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     2461 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     2521 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     2581 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatga aggccttgac
     2641 aggatatatt ggcgggtaaa ctaagtcgct gtatgtgttt gtttgagatc tcatgtgagc
     2701 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2761 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2821 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2881 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2941 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3001 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3061 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3121 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3181 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaag
     3241 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3301 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3361 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3421 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3481 aagtatatat gtgtaacatt ggtctagtga ttagaaaaac tcatcgagca tcaaatgaaa
     3541 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     3601 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     3661 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     3721 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     3781 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     3841 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     3901 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     3961 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     4021 tgggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     4081 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     4141 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     4201 tcggtagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     4261 atcagcatcc atgttggaat ttaatcgcgg ccttgagcaa gacgtttccc gttgaatatg
     4321 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga
     4381 tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttgttg
     4441 aataaatcga acttttgctg agttgaagga tcagatcacg catcttcccg acaacgcaga
     4501 ccgttccgtg gcaaagcaaa agttcaaaat caccaactgg tccacctaca acaaagctct
     4561 catcaaccgt ggctccctca ctttctggct ggatgatggg gcgattcagg cgatccccat
     4621 ccaacagccc gccgtcgagc gggctttttt atccccggaa gcctgtggat agagggtagt
     4681 tatccacgtg aaaccgctaa tgccccgcaa agccttgatt cacggggctt tccggcccgc
     4741 tccaaaaact atccacgtga aatcgctaat cagggtacgt gaaatcgcta atcggagtac
     4801 gtgaaatcgc taataaggtc acgtgaaatc gctaatcaaa aaggcacgtg agaacgctaa
     4861 tagccctttc agatcaacag cttgcaaaca cccctcgctc cggcaagtag ttacagcaag
     4921 tagtatgttc aattagcttt tcaattatga atatatatat caattattgg tcgcccttgg
     4981 cttgtggaca atgcgctacg cgcaccggct ccgcccgtgg acaaccgcaa gcggttgccc
     5041 accgtcgagc gcctttgccc acaacccggc ggccggccgc aacagatcgt tttataaatt
     5101 tttttttttg aaaaagaaaa agcccgaaag gcggcaacct ctcgggcttc tggatttccg
     5161 atccccggaa ttagatcttg gcaggatata ttgtggtgta acgttaacat taacgcttac
     5221 aatttccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     5281 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     5341 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac
     5401 tcactatagg gcgaattggg taccgggccc cccctcgagg tcgacggtat cgata
//
